Alzheimer's disease (AD) is a degenerative brain disorder characterized clinically by progressive loss of memory, cognition, reasoning, judgment and emotional stability that gradually leads to profound mental deterioration and ultimately death. AD is a very common cause of progressive mental failure (dementia) in aged humans and is believed to represent the fourth most common medical cause of death in the United States. AD has been observed in all races and ethnic groups worldwide and presents a major present and future public health problem.
In Germany about 65,000 cases of AD are newly diagnosed every year. The disease is currently estimated to affect about two to three million individuals in the United States alone. AD is at present incurable. No treatment that effectively prevents AD or reverses its symptoms or course is currently known.
The brains of individuals with AD exhibit characteristic lesions termed senile plaques, and neurofibrillary tangles. Large numbers of these lesions are generally found in several areas of the human brain important for memory and cognitive function in patients with AD. Smaller numbers of these lesions in a more restricted anatomical distribution are sometimes found in the brains of aged humans who do not have clinical AD. Senile plaques and amyloid angiopathy also characterize the brains of individuals beyond a certain age with Trisomy 21 (Down's Syndrome) and Hereditary Cerebral Hemorrhage with Amyloidosis of the Dutch-Type (HCHWA-D).
At present, a definitive diagnosis of AD usually requires observing the aforementioned lesions in the brain tissue of patients who have died with the disease or, rarely, in small biopsied samples of brain tissue taken during an invasive neurosurgical procedure. The principal chemical constituent of the senile plaques and vascular amyloid deposits (amyloid angiopathy) characteristic of AD and the other disorders mentioned above is an approximately 4.2 kilodalton (kD) protein of about 3943 amino acids originally designated the amyloid-β peptide (Aβ) or sometimes βAP, AβP or β/A4. Nowadays the nomenclature Aβ is generally accepted to describe this polypeptide.
Aβ was first purified and a partial amino acid sequence reported in Glenner, G. G., and Wong, C. W., Biochem. Biophys. Res. Commun. 120 (1984) 885-890. The isolation procedure and the sequence data for the first 28 amino acids are described in U.S. Pat. No. 4,666,829. Forms of Aβ having amino acids beyond number 40 were first reported by Kang, J., et al., Nature 325 (1987) 733-736.
Roher, A. E., et al., Proc. Natl. Acad. Sci. USA 90 (1993) 10836-10840 showed that Aβ(142) is the major constituent in neuritic plaques (90%) with significant amounts of isomerized and racemized aspartyl residues. The authors also showed that Aβ(17-42) also predominates in diffuse plaques (70%), while Aβ(1-40) is the major constituent in the meningovascular plaques, comprising 60% of the total Aβand, in parenchymal vessel deposits Aβ(1-42) represents 75% of the total Aβ.
Iwatsubo, T., et al., Neuron 13 (1994) 45-53 showed that Aβ 42(43)-positive senile plaques are the major species of Aβ in sporadic Aβ brain.
Molecular biological and protein chemical analyses conducted during the last several years have show that Aβ is a small fragment of a much larger precursor protein, referred to as the amyloid precursor protein (AβP), that is normally produced by cells in many tissues of various animals, including humans. Knowledge of the structure of the gene encoding APP has demonstrated that Aβarises as a peptide fragment that is cleaved from the carboxy-terminal end of APP by a set of enzymes termed α-, β-, and γ-secretases. The precise biochemical mechanism by which the Aβ fragment is cleaved from APP and subsequently deposited as amyloid plaques in the cerebral tissue and in the walls of cerebral and meningeal blood vessels is currently unknown.
Several lines of evidence indicate that progressive cerebral deposition of Aβ plays a seminal role in the pathogenesis of Aβ and can precede cognitive symptoms by years or decades (for review, see Selkoe, D. J., J. Neuropath. and Exp. Neurol. 53 (1994) 438-447; and Selkoe, D. J., Neuron 6 (1991) 487).
Despite the progress, which has been made in understanding the underlying mechanisms of AD, there remains a need to develop methods for use in diagnosis of the disease.
Numerous biochemical electron microscopic and immunochemical studies have reported that Aβ is highly insoluble in physiologic solutions at normal pH. See, for example, Glenner, G. G., and Wong, C. W., Biochem. Biophys. Res. Commun. 122 (1984) 1131-1135; Masters, C. L., et al., Proc. Natl. Acad. Sci. USA 82 (1985) 4245-4249; Selkoe, D. J., et al., J. Neurochem. 46 (1986) 1820-1834.
Furthermore, this insolubility was predicted by and is consistent with the amino acid sequence of Aβ which includes a stretch of hydrophobic amino acids that constitutes part of the region that anchors the parent protein (APP) in the lipid membranes of cells. Hydrophobic, lipid-anchoring proteins such as the Aβ-part of APP are predicted to remain associated with cellular membranes or membrane fragments and thus not to be present in physiologic extracellular fluids. The aforementioned studies and many others have reported the insolubility in physiologic solution of native Aβ purified from AD brain amyloid deposits or of synthetic peptides containing the Aβ sequence. The extraction of Aβ from cerebral amyloid deposits and its subsequent solubilization has required the use of strong, non-physiologic solvents and denaturants.
Physiologic, buffered salt solutions that mimic the extracellular fluids of human tissues have uniformly failed to solubilize Aβ.
Immunoassays in general are performed at physiological pH. Polypeptides soluble at physiological buffer conditions, therefore, are extensively used in various immunoassay methods, such as, e.g., ELISA (enzyme-linked immunosorbent assay), for example in diagnosis of and screening for a certain disease.
Due to its insolubility under physiological buffer conditions, and due to its sticky nature the Aβ peptide is difficult to use in an immunoassay. E.g., in a sandwich assay format Aβ is difficult to handle, because it tends to aggregate or even precipitate. Due to its sticky nature Aβ may also lead to false results caused by unspecific binding.
Although it is possible to solubilize Aβ by means of strongly chaotropic reagents or appropriate detergents, the material solubilized in such a manner is of limited use as a diagnostic tool.
The insolubility of Aβ at physiological buffer conditions in addition renders this protein a very difficult target of routine (bio-) chemical procedures. The vast majority of “labeling chemistries”, i.e., the chemical procedures used for binding a label, e.g., a marker group to a polypeptide, is based on nucleophilic chemistry and thus rather restricted to a pH window from about pH 6 to about pH 8 and thus only works at more or less physiological buffer conditions. These routine procedures, e.g., as described in Aslam, M. and Dent, A., The preparation of protein—protein conjugates in “Bioconjugation”, eds. M. Aslam and A. Dent, McMillan Reference, London (1998), pp. 216-363, either do not work properly or are difficult to carry out with the Aβ peptide.
Therefore a tremendous need exists to provide Aβ in a readily soluble form, a form in which Aβ is, e.g., soluble at physiological pH, stable in solution and/or convenient to produce and/or handle.
It was the task of the present invention to investigate whether Aβ can be provided in a form, which is readily soluble at physiological buffer conditions.
We found that folding helpers, e.g., many members of the peptidyl prolyl isomerase (PPI) class, especially from the FKBP family, not only exhibit catalytic activity, but also bring about drastic beneficial effects on solubility of amyloidogenic proteins, or more generally speaking, of proteins tending to aggregation, like the Aβ peptide. They do so by forming soluble complexes with such proteins that are otherwise (i.e. in an unchaperoned, isolated form) prone to aggregation. Aβ which is hardly soluble or insoluble under physiological conditions turned out to be soluble under mild physiological conditions (i.e. without need for solubilizing additives such as detergents or chaotropic agents) once it is present in form of a complex with an appropriate PPI chaperone. Thus, we were able to produce, for example, soluble Aβ-chaperone complexes comprising, e.g., the Aβ(1-42) peptide (otherwise an aggregation prone) protein and SlyD, FkpA or other FKBPs as solubility-conferring chaperones.
A soluble complex comprising an Aβ peptide and a PPI-class chaperone can for example be obtained from a single recombinant protein comprising both an Aβ peptide and a PPI class chaperone. A recombinant protein comprising Aβ and a chaperone selected from the peptidyl-prolyl-isomerase class of chaperones is described.
Most intriguingly, a fusion protein comprising both an Aβ peptide and a PPI chaperone can be solubilized and renatured easily and has been found to form a soluble intramolecular Aβ-chaperone complex that enables e.g., the convenient labeling of said complex.
It is now possible to provide an Aβ in a readily soluble form for use as standard material in immunoassays. It is also possible to produce a labeled chaperone-Aβ complex wherein solely the chaperone is labeled, making sure that the Aβ-antigen is not modified or negatively influenced (e.g. in terms of conformation) by such labeling.
The Aβ-chaperone complexes we describe here provide a convenient means to produce a soluble labeled Aβ peptide for immunoassays irrespective of the detection format used.
The novel complexes comprising Aβ and SlyD, for example, are readily soluble, e.g., under physiological conditions, they can be easily labeled in convenient pH ranges, and they can be used to great advantage in the detection of Aβ by immunological techniques or for immunization.